Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Background:The oxytocin receptor couples to multiple G proteins, leading to different physiological responses. Results: We screened for functional selective oxytocin receptor agonists and identified two analogs that activate individual G i subunits. Conclusion: Functional selective analogs discriminate among different receptor conformations coupled to G i proteins. Significance: These compounds will contribute to the development of selective drugs with new selectivity and therapeutic profiles.

Background: GHS-R1a activates multiple signaling pathways mediating feeding and addictive behaviors. Results: Some GHS-R1a ligands activate G q but not G i/o and fail to recruit ␤-arrestin2; others act as selective inverse agonists at G q compared with G 13 . Conclusion: Synthetic ligands can selectively activate or reverse G q -dependent signaling at GHS-R1a. Significance: Ligand-biased signaling can be exploited for the development of selective drugs to treat GHS-R1a-mediated disorders.

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Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands.

The role of β-adrenergic receptors (β-ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in β-AR-dependent control of proliferation and adhesion of nontumor human breast cell line MCF-10A. Low concentrations of a β-agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p-Erk1/2)-dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2-site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08±3.09 nM), while ISO significantly inhibited p-Erk1/2 according to a 1-site model (EC50: 0.25±0.13 nM). Using β-AR-selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage-dependent signaling in which low ISO concentrations target a β2-AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/adhesion-signaling module, while higher concentrations engage a concomitant activation of another β2-AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA-dependent signaling module. Our data provide a new molecular basis for the dose-dependent switch of β-AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-m-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.The fusion induced by human immunodeficiency virus type 1 (HIV-1) 6 is a multistep process that requires for the virus to bind to two different host cell surface receptors. Current models suggest an initial interaction between the trimeric viral envelope protein gp120 and the primary CD4 receptor on target cells. This results in a conformational change of gp120 that exposes a co-receptor binding site. After binding to the co-receptor molecule, either CXCR4 or CCR5 depending on virus tropism, additional structural changes take place, allowing the viral gp41 protein to initiate the fusion of viral and cellular membranes. This last step leads to the release of the viral genome into the cytoplasm of the target cell (for a review, see Ref. 1).A more efficient co-immunoprecipitation of CD4 with CCR5 compared with CXCR4 supports the existence of preferential interactions between CCR5 and CD4 (2), which could be at the origin of the predominance of R5-tropic HIV. In addition, a multiplicity of interactions are involved, since the attachment of several gp120 envelope trimers to multiple CD4 (3) and to multiple CCR5 co-receptors (4) seems to be needed for infection to proceed (5).Thus, the fusion process involves the encounter and interaction of multiple receptors at the plasma membrane. A compartmentalization of the partners could favor this step of the infection. The virus may take advantage of a clustering to rapidly form multivalent interactions with the receptors and co-receptors required for fusion. The heterogeneous patchiness of the membrane can account for such an organization. Indeed, the current view of the plasma membrane is a "dynamically structured mosaic model" (6) characterized by a dynamic organization into domains scaling from the nanometer to micrometer range (7,8). Evidence is accumulating that compartmentalization of membrane components could be essential for the regulation of cellular functions (7).In the case of CD4 and CCR5, several results suggest the existence of such a compartmentalization. First, electron microscopy observations show that CCR5, CXCR4, and CD4 form homogeneous m...

The degeneracy of the guanine radical cation, which is formed in DNA by oxidation of guanine by electron transfer, was studied by a detailed analysis of the oxidation products of guanine on oligonucleotide duplexes and by labeling experiments. It was shown that imidazolone, the major product of guanine oxidation, is formed through a one-electron oxidation process and incorporates one oxygen atom from O2. The formation of 8-oxo-7,8-dihydroguanine by a two-electron oxidation process was a minor pathway. The two-electron oxidation mechanism was also evidenced by the formation of a tris(hydroxymethyl)aminomethane adduct.